Project Summary. This K99/R00 proposal concerns the structure, function, and regulation of key proteins involved in cell signaling, PTEN and USP7. PTEN is a tumor suppressor lipid phosphatase that catalyzes the removal of the 3'-phosphate from the membrane phospholipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. Since PIP3 is a key regulator of cell growth and insulin signaling, it is imperative that PTEN activity be tightly controlled. Loss of function PTEN mutations are frequently observed in cancer. PTEN is post-translationally regulated by N-terminal ubiquitination and C-terminal phosphorylation but the detailed structural and mechanistic impacts of these post-translational modifications (PTMs) are not well understood. USP7 is a Cys hydrolase that is a deubiquitinase (DUB), catalyzing the cleavage of the ubiquitin/lysine isopeptide bond. USP7's ubiquitinated protein substrates include PTEN and MDM2. Deubiquitination of PTEN is reported to inhibit its translocation from the cytosol to the nucleus. Moreover, USP7 has been shown to enhance the cellular stability of MDM2, and this is important because MDM2 is an E3 ubiquitin ligase for major tumor suppressor protein p53. It is unclear what molecular features drive USP7's substrate selectively and how it is regulated in the cell. USP7 is modified on both its N- and C-termini by phosphorylation and acetylation but the regulatory roles of these PTMs are unclear. Here, we will address how PTEN and USP7 are regulated by PTMs using new and emerging semi-synthetic approaches. These semi-synthetic methods can facilitate site-specific and stoichiometric installation of PTMs and their mimics into PTEN and USP7. Aim 1 seeks to define the molecular basis for PTEN regulation by C-terminal tail phosphorylation using structural approaches. Conformational closure of PTEN is driven by phosphorylation of its C-terminal tail at positions 380, 382, 383, and 385 resulting in an inhibited enzyme, reduced plasma membrane binding, and increased stability. This aim employs biomolecular NMR, crystallography, and mutagenesis to understand the structural and mechanistic basis for conformational closure. Aim 2 will employ a series of biochemical and cellular methods to define the function of Lys13 monoubiquitination, enhancing our understanding of how this PTM may promote the shuttling of PTEN from the cytosol to the nucleus. Aim 3 will address what molecular features drive USP7?s substrate selectivity and how USP7 PTMs (Ser18, Tyr1091, Thr1092, and Tyr1093 phosphorylation; Lys1096 acetylation and ubiquitination) regulate its function. Overall, these proposed studies can greatly enhance our understanding of the function and regulation of PTEN and USP7 which can spotlight possible targets for therapy. In addition, this proposal can also increase the PI's breadth of scientific skills and experiences as he seeks to chart a course for an independent academic career.